Method and Assembly for Dna Isolation With Dry Reagents

ABSTRACT

A DNA isolation method by removal of constituents, interfering with a subsequent PCR is disclosed. According to an embodiment of the method, all substances and method steps are fully integrated into a closed unit (cartridge) for single use, which allows entry of a DNA-containing sample and DNA-binding substrates are used for isolating the released DNA. In particular, when the method is applied to DNA isolation from whole blood by disruption of white blood cells, the reagents, required for carrying out the cell disruption and other reactions, are stored in a form which is stable at room temperature. For disrupting the white blood cells and for DNA isolation, a dry stored lysis reagent is dissolved in an aqueous solution and brought into contact with the white blood cells. The corresponding assembly includes a unit, for housing DNA-containing biological containers and/or reagents, whereby at least one microchannel is provided to contain reagents, whereby the reagent is present in the microchannel as a dry mixture with a negligible vapour pressure, which forms a stable substance at room temperature.

PRIORITY STATEMENT

This application is the national phase under 35 U.S.C. §371 of PCTInternational Application No. PCT/EP2005/051941 which has anInternational filing date of Apr. 28, 2005, which designated the UnitedStates of America and which claims priority on German Patent Applicationnumber 10 2004 021 780.7 filed Apr. 30, 2004, the entire contents ofwhich are hereby incorporated herein by reference.

FIELD

The invention generally relates to a method for DNA isolation with dryreagents. The invention also generally relates to an associated assemblyfor carrying out the method.

BACKGROUND

For nucleic acid analysis to answer human genomic questions, e.g. inwhite blood cells from whole blood, the cells must firstly be broken upin a sample preparation step and the DNAs thereby released mustsubsequently be isolated. It is in this case necessary to remove bloodconstituents such as hemoglobin, immunoglobulins and lactoferrin, whichcould inhibit a subsequent PCR.

In the laboratory, these working steps are carried out according to asufficiently well-known prior art. Besides other methods, for example,cells can be broken up (so-called lysis) with an alkaline solution(NaOH) and the DNAs can subsequently be bound to silica-coated magnetbeads. By applying a magnetic field, the magnet beads laden with DNA arefixed and washed. The DNA isolated in this way can subsequently be usedwith or without beads for a PCR (Polymerase Chain Reaction).

In the prior art, the DNA-binding magnet beads are used as a suspensionin a cell lysis buffer. All the working steps are carried out forexample in 1.5 ml reaction vessels (so-called “Eppendorf” vessels). Forexample, 10 μl of whole blood is added to a predetermined volume of thebead suspension (e.g. 200 μl). The blood cells are thereby disintegratedand the DNAs are released. The magnet beads bind the DNA and form aDNA/bead complex. Such a DNA/bead complex can subsequently be fixed by amagnetic field on the vessel wall of the Eppendorf tuber, so thatwashing steps can be carried out to remove PCR-inhibiting substances.The PCR can subsequently be carried out.

Conduct of the latter method is contingent on the provision oflaboratory equipment such as the “Eppendorf” vessels (tubes), tubeholding devices including magnets, pipetting equipment, coolingcontainers for reagents, and must be carried out by trained personnelwhile complying with safety rules (infection risk, waste disposal,etc.). A plurality of volumetric and highly accurate dosings ofsubstances which are sometimes hazardous to health (e.g. NaOH) first becarried out by pipetting. These working steps are also time-consuming.

US 2002/0022261 A1 has already disclosed systems for miniaturizedgenetic analysis and associated operating methods, in which a cartridgewith at least one input and a porous passage, which is intended to haveDNA-binding properties, is used. Disintegration of cells takes place, towhich end reagents are provided optionally on the vessel wall.Structured regions of the wall may furthermore be coated withDNA-binding materials, optionally also magnet beads. Overall a series ofdifferent proposals are provided there in order to perform themeasurement for the genetic analysis, although a continuous method isstill not carried out. A mixture for the isolation of DNA is furthermoreknown from EP 0 723 549 B1, this mixture especially containing silicagel, glass particles and at least one chaotropic salt.

Furthermore, WO 99/33559 A describes a device for separating and analytefrom a fluid sample, which comprises the cartridge with a lysationchamber in the sample flow path, the cells of the sample being filteredout via a filter and disintegrated. The analyte separated in this way isfed to individual sensor chambers in the device for analysis.

SUMMARY

In at least one embodiment of the invention, the PCR is performedentirely in an integrated miniaturized cartridge and in at least oneembodiment, an associated assembly is provided.

In the scope of at least one embodiment of the invention, a methodincludes disintegration of biological structures containing DNA, forexample cells, bacteria or viruses. It is therefore possible to studywhole blood samples in respect of DNA information in a particularlyadvantageous

Besides the above prior art, at least one embodiment of the invention isalso based on WO 02/0072262 A1 entitled “Analysis Device”. This hasalready described the use of dryly stored, room-temperature stablereagents in microchannels or microcavities of a “chip card”, which areput into solution by supplying water shortly before use. This prior artentails providing the dry reagents in a pre-portioned form, so that aquantitative analysis medium is obtained after dissolving. At least oneembodiment of the present invention on the other hand, involves celldisintegration of a biological structure, for example the isolation ofDNA from a whole blood sample for the purpose of subsequent PCR and/oranalysis, the DNAs isolated from the sample being prepared-in a suitableway.

At least one of the following advantages, compared with the previouslyused laboratory method, are obtained with the method according to atleast one embodiment of the invention:

-   all the materials and methods are fully integrated in a closed    single-use cartridge;-   it ensures that the reagents are held in a secure form not hazardous    to health, which is stable when stored at room temperature;-   no manual working steps are necessary, other than injection of the    blood sample;-   no direct contact takes place with materials that are hazardous to    health, i.e. blood and reagent waste remain in the cartridge; the    cartridge is small inexpensive to make in mass production.

The assembly according to at least one embodiment of the inventionincludes at least one of the following features:

-   at least one microchannel or microcavity is provided. In the    microchannel or the microcavity, the lysis medium is applied-   in particular with the inclusion of the film-forming agent with the    substrate for the DNA on the wall of the channel.

A lysis medium introduced into the microchannel or microcavity has thefollowing properties:

-   lysis properties for white blood cells and/or other cells, bacteria,    viruses;-   solid, or liquid with a negligible vapor pressure;-   stable at room temperature;-   good adhesion to microchannel or microcavity walls.

In at least one embodiment, in a continuous method sequence, the DNAcontained in the sample is released, collected as isolated DNA andsuitably brought to the site of the PCR or a detection.

BRIEF DESCRIPTION OF THE DRAWINGS

Further details and advantages of the invention will be found in thefollowing figure description of example embodiments with the aid of thedrawings in conjunction with the patent claims.

FIG. 1 shows the plan view of an analysis device (cartridge) and

FIG. 2 to FIG. 6 respectively show a detail of FIG. 1 in longitudinalsection along the line II-II to illustrate the DNA isolation from wholeblood, the individual FIGS. 2 to 6 respectively containing differentfunction steps.

DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS

Samples containing DNA will be defined in the following description.Such samples may be solutions of DNA in a liquid, but alsoDNA-containing suspensions of biological structures. Cells, bacteria orviruses, for example, are to be understood as biological structures inthe present context. Disintegration of the biological structure is thennecessary in order to release the DNA. If whole blood samples are usedaccording to a human genomic task, for example, then cell disintegrationof white blood cells in which the DNA is localized is necessary.

FIG. 1 represents an analysis device, which also as “cartridge” belowand may be designed as a central or decentral measuring device. Inparticular, the analysis device is designed in the manner of a chip card(“lab-on-a-chip”), which contains all the aspects for treating andevaluating measurement samples. An associated control/readout devicenecessary for proper operation of the device is not shown in thiscontext.

In detail the device includes a card-like body 1 made of plastic, whichincludes inlets and outlets for fluids. In particular, an inlet (“port”)2 is provided for introducing water and an inlet 3 (“port”) is providedfor introducing a measurement sample, for example blood. What isessential is that measurement samples and solvents are transported andcombined via suitable fluidic devices 2 to 10, and in particularchannels and cavities of different geometry.

Besides the aforementioned water and sample ports 2, 3, the fluidicdevices specifically contain two reagent channels 4, 4′ as well as aflow channel 5 with an outlet 6, a reception channel 8 for waste and afurther fluidic channel 9. A central mixing region in the flow channel 5for the sample preparation is denoted by 10.

After treatment of the sample in the mixing section 10 and afterisolation of the DNA contained in the measurement sample, the releasedDNAs are collected and fed to a PCR chamber 20 for carrying out a PCR(Polymerase Chain Reaction).

A process for carrying out PCR especially with dry reagents in anassembly according to FIG. 1 is described in detail in the parallelpatent application in the name of the Applicant with the sameapplication priority entitled “PCR process and arrangement for DNAamplification using dry reagents”.

Besides the PCR means, the card 1 (“cartridge”) furthermore contains adetection module 30 with a feed 32 and discharge 31, as well asassociated connections for sensor signal readout. Means for receivingreagents for the detection, for example channels 4 and 4′, as well asfor receiving waste such as blood and consumed reagent solutions, forexample channels 8 and 9, are furthermore provided. This ensures anintegration in which no substances hazardous to health can escape.

It can be seen from FIGS. 2 to 6 that the plastic card 1 of FIG. 1 witha water port 2 and a blood port 3 contains a flow channel 5 via whichwater as a washing liquid, and for example a blood or DNA solution or acell/bacteria/virus suspension as a measurement sample, are introducedinto the otherwise closed system. The waste is transferred via an outlet6 into the waste channel 8, or into a waste cavity. On the opposite sidefrom the plastic card 1, the flow channel 5 is covered with an adhesivefilm 7. In the central region of the flow channel 5, a mixing section 10is formed in which the measurement samples are prepared especially forDNA isolation from whole blood.

The DNA isolation from whole blood takes place via disintegration of thewhite blood cells. To this end, the cells are broken up chemically witha lysis reagent and the DNA contained in them is released and collected.In particular, the known procedure is employed in which the DNA is boundat least temporarily via so-called magnet beads after disintegration ofthe cell, and is concentrated by an external magnetic field.

It can be seen especially in FIG. 2 that a storage-stable dry substance11, which is water-soluble, is arranged in a region 10 of the flowchannel S in the plastic card 1. Storage stability in this context isintended to mean that the solid can be stored for several months at roomtemperature while preserving the property of inducing the sampledisintegration.

The dry substance 11 exhibits good adhesion to the walls of the flowchannel in FIGS. 2 and 3. This may, for example, be achieved by adding afilm-forming agent. To this end, specifically, a mixture 11 of a knownlysis reagent with magnet beads 13 is arranged over a large area in theregion of the mixing section 10. A magnet 15 is furthermore symbolicallyindicated, which illustrates the magnetic treatment to concentrate themagnet beads 13 with DNA bound on them.

It can be seen from FIG. 3 and FIG. 4 that whole blood 12 is introducedinto the measurement system via a pipette tip 17 in the arrangementaccording to FIG. 2. The port 3 is closed by way of an adhesive film 18after the sample has been introduced.

Water or a buffer solution is introduced via the port 2. Water or buffersolution mixes with the blood sample 12 along the mixing section, thelysis reagent 11 together with the magnet beads 13 likewise beingdissolved or suspended, and activated. Accordingly, the cell walls ofthe white blood cells are broken up by the lysis reagent and the DNAsthereby released are bound to the surface of the magnet beads.

According to FIG. 4, the water/the buffer solution thus dilutes theblood sample and simultaneously dissolves the lysis reagent, whichserves to ensure disintegration of the white blood cells. Likewise,moreover, the magnet beads of the mixture 11 which lie on the wall ofthe channel are brought into solution or suspended, the DNAs being boundvia these magnet beads 13. In this way, the following essential methodsteps, specifically:

-   sample dilution,-   reagent dissolving/magnet bead suspension,-   cell disintegration and-   DNA binding    are carried out in a single process.

The latter is illustrated with the aid of FIG. 5. The DNA magneticallytreatable via the magnet beads 13 can now be collected, whilePCR-inhibiting substances are washed out by water or a buffer solutionand discharged via the outlet 6.

As a result of the measures described above, according to FIG. 6 the DNAbound via the magnet beads 13 is concentrated on one of the magnet polesof the magnet 15. This DNA can be used for an analysis, a PCR inparticular being carried out first.

As an alternative to employing mobile i.e. suspended magnet beads incombination with a magnetic field as a DNA-binder(s), it is alsopossible to use immobilized i.e. static DNA-binder(s), placed at theoutput of the cell disintegration channel. In this embodiment, the DNAreleased from the cells is extracted from the liquid stream when itleaves the cell disintegration channel by flowing over the staticDNA-binder(s), and is bound on the DNA-binder(s). For example, hydrogelswith DNA-binding capture molecules or the like may be envisaged asDNA-binder(s).

The method according to an embodiment of the invention and theassociated assembly provide in particular one-stage, simple and rapidDNA isolation, which necessitates only a minimum of microfluidicdevices, reagents and/or method steps.

The isolated DNA can subsequently be subjected to the PCR. PCR increasesthe concentration of the DNA to an analytically detectable value. ThePCR can now be incorporated into the analysis process. The analysis thentakes place in the detection module 30 according to FIG. 1, which willnot be discussed in further detail here.

In summary, at least one embodiment of the invention implements thefollowing measures:

-   the substrates introduced in the microchannel or microcavity have    DNA-binding properties. To this end, for example, DNA-binding magnet    beads may be used;-   the lysis reagents and magnet beads are contained in particular    together in a single dry matrix;-   an input port is provided for a whole blood sample    cell/bacteria/virus suspension);-   at least one way for supplying water is provided. This may, for    example, be a feed port for connection to an external water supply;-   at least one way/device is optionally provided for admixing salts,    for example in order to adjust a defined ionic strength;-   at least one way/device (channels and cavities) is optionally    provided for diluting the blood sample;-   optionally further ways/devices, for example channels and cavities,    are provided in order to prepare defined salt/buffer solutions for    washing the bound DNA, e.g. DNA-magnet bead complex;-   at least one way/device is provided, preferably outside the    cartridge, so that blood or a blood/water, blood/buffer mixture can    flow through the microchannel or microcavity coated with lysis/bead    reagent;-   at least one way/device is provided, for example at the output of    the microchannel, for generating a magnetic field to fix the    DNA/magnet bead complex in the PCR cavity, these ways/devices    preferably lying outside the cartridge.

This ensures that the entire analysis process, including the samplepreparation, takes place in the closed system constituted by adisposable cartridge made of environmentally friendly plastic.

Example embodiments being thus described, it will be obvious that thesame may be varied in many ways. Such variations are not to be regardedas a departure from the spirit and scope of the present invention, andall such modifications as would be obvious to one skilled in the art areintended to be included within the scope of the following claims.

1. A method for DNA isolation while removing constituents that interferewith a subsequent PCR, the method comprising: integrating materials in aclosed analysis single-use unit which allows a sample with DNA to enter;using dry reagents to release the DNA, stored in the unit in a form thatis stable at room temperature; using DNA-binding substrates to isolatethe released DNA; bringing a dry matrix, lying on the wall in adisintegration channel and including lysis reagents and the DNA-bindingsubstrates, in contact with biological structures, to disintegrate thebiological structures containing the DNA, the DNAs released by contactwith the lysis reagents being bound directly to the DNA-bindingsubstrates; sand collecting the substrates with the DNAs bound thereonand transporting the collected substrates a site of the PCR.
 2. Themethod as claimed in claim 1, wherein white blood cells are used asstructures containing DNA.
 3. The method as claimed in claim 2, whereinthe dryly stored and storage-stable lysis reagent is brought in contactwith the white blood cells with the aid of water.
 4. The method asclaimed in claim 1, wherein diluting of the sample, dissolving orsuspending of the dry reagent, disintegrating of the biologicalstructures and binding of the DNA take place in a single step.
 5. Themethod as claimed in claim 1, wherein magnet beads are used as theDNA-binding substrate.
 6. The method as claimed in claim 1, wherein ahydrogel with DNA capture substances, immobilized at the exit of thecell disintegration channel, is used as the DNA binding substrate. 7.The method as claimed in claim 6, wherein the released DNA is bound andtransported by the magnet beads to a at least one of a collection siteand a reaction site.
 8. The method as claimed in claim 5, wherein themagnet beads are concentrated by applying a magnetic field.
 9. Themethod as claimed in claim 8, wherein flowing magnet beads areconcentrated by a static magnetic field.
 10. The method as claimed inclaim 1, wherein the PCR is carried out by thermocycling with drylystored water-soluble reagents at the reaction site.
 11. The method asclaimed in claim 1, wherein the blood sample put into the unit isdiluted and pumped through the reagent channel to the reaction channelfor the PCR.
 12. The method as claimed in claim 11, wherein residualblood, blood constituents and the reagent waste remain in the unit afterthe DNA isolation, so that no direct contact can take place withsubstances hazardous to health.
 13. The method as claimed in claim 1,wherein a disposable product, which is small and inexpensive to make inmass production, is used as the single-use unit in which the reactionsare performed.
 14. An assembly for DNA isolation while removingconstituents that interfere with a subsequent PCR comprising: a unit forreceiving at least one of samples and reagents, including at least onemicrochannel for receiving reagents; and a dry matrix including lysisreagent and DNA-binding substrate, provided in the microchannel as amixture with a negligible vapor pressure, the dry matrix forming astable substance at room temperature, wherein the mixture of lysisreagent and the DNA-binding substrate is bound as a layer over a largearea on the wall of the microchannel by adding a film-forming agent,wherein the dry matrix is adapted to be brought into contact withbiological structures to disintegrate the biological structurescontaining DNA, the DNA released by contact with the lysis reagentsbeing bound directly to the DNA-binding substrate, and the substrate,with the DNA bound thereon, being adapted to be collected andtransported to a site of the PCR.
 15. The assembly as claimed in claim14, wherein the biological structures containing DNA are in at least oneof particular cells, bacteria, and viruses.
 16. The assembly as claimedin claim 15, wherein, for carrying out the disintegration of abiological structure, the dry substance with a negligible vapor pressurein the microchannel is a lysis reagent which has specific properties forwhite blood cells.
 17. The assembly as claimed in claim 16, wherein thechannel for the lysis substance opens in a PCR cavity.
 18. The assemblyas claimed in claim 17, wherein, for carrying out the disintegration ofa biological structure, the dry substance with a negligible vaporpressure in the microchannel is a lysis reagent which has specificproperties for white blood cells.
 19. The assembly as claimed in claim18, wherein the substrates are DNA-binding magnet beads.
 20. Theassembly as claimed in claim 19, wherein substrates, includingDNA-binding properties, are provided in at least one of the microchanneland the microcavity.
 21. The assembly as claimed in claim 14, furthercomprising an input port for whole blood samples.
 22. The assembly asclaimed in claim 14, further comprising: means for supplying water. 23.The assembly as claimed in claim 14, further comprising dry buffersubstances for adjusting a defined ionic strength.
 24. The assembly asclaimed in claim 14, further comprising: means for mixing a blood sampleand at least one of water and, a buffer solution.
 25. The assembly asclaimed in claim 14, further comprising: means for permitting at leastone of blood, blood/water and blood/buffer mixtures to flow through atleast one of the microchannel and or microcavity coated with reagent.26. The assembly as claimed in claim 14, further comprising: means forgenerating a magnetic field for the purpose of fixing a DNA-magnet beadcomplex in the PCR cavity.
 27. The assembly as claimed in claim 14,further comprising: means for thermocycling.
 28. (canceled) 29.(canceled)
 30. The assembly as claimed in claim 21, further comprising:means for supplying water.
 31. The assembly as claimed in claim 30,further comprising dry buffer substances for adjusting a defined ionicstrength.
 32. The assembly as claimed in claim 31, further comprising:means for mixing a blood sample and at least one of water and a buffersolution.
 33. The assembly as claimed in claim 32, further comprising:means for permitting at least one of blood, blood/water and blood/buffermixtures to flow through at least one of the microchannel andmicrocavity coated with reagent.
 34. The assembly as claimed in claim33, further comprising: means for generating a magnetic field for thepurpose of fixing a DNA-magnet bead complex in the PCR cavity.
 35. Theassembly as claimed in claim 34, further comprising: means forthermocycling.